Modulation of pro-inflammatory gene expression by nuclear lysophosphatidic acid receptor type-1

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.     

Proinflammatory gene induction by platelet-activating factor mediated via its cognate nuclear receptor

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H(2)O(2). Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-kappaB binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-kappaB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.     

The East Flanders Prospective Twin Survey (EFPTS).

The East Flanders Prospective Twin Survey (EFPTS) is a registry of multiple births in the province of East-Flanders, Belgium. It has several unique features: it is population-based and prospective, with the possibility of long-term follow-up; the twins (and higher order multiple births) are ascertained at birth; basic perinatal data are recorded; chorion type and zygosity are established; and since 1969 placental biopsies have been taken and frozen at -20 degrees C for later determination of genetic markers. The EFPTS is the only large register that includes placental data and allows differentiation of three subtypes of monozygotic (MZ) twins based on the time of the initial zygotic division: the dichorionic-diamnionic pairs (early, before the fourth day after fertilisation), the monochorionic-diamnionic pairs (intermediate, between the fourth and the seventh day post fertilisation), and the monochorionic-monoamnionic pairs (late, after the eight day post fertilisation). This added a new dimension to didymology (the science of twins; delta iota delta upsilon mu omicron zeta = twins): the timing of twinning. Studies can be initiated on primary biases, those originating "in utero". Such studies may throw new light on the controversy over the validity of the classic twin method, the consequences of early embryological events (before and just after implantation of the embryo), the origin of congenital malformations, the sex proportion of multiples, the gene-environment interactions as far as intrauterine environment is concerned, to name but a few.     

Isoxazolin-5-one derivatives in Lathyrus odoratus during development and growth

Variations in the concentrations of isoxazolin-5-ones and of some non-protein amino acids in the dry seeds, seedlings and various parts of mature Lathyrus odoratus plants were examined. The lathyrogenic compounds α-amino-γ-(isoxazolin-5-on-2-yl)-butyric acid, 2-cyanoethyl-isoxazolin -5-one and γ-glutamyl-β-aminopropionitrile were major products during development and growth.     

CRF stimulates α-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells

Ovine corticotropin-releasing factor (CRF) stimulates α-MSH release and cyclic AMP accumulation in rat pars intermedia cells in culture at ED₅₀ values of 1 and 6 nM, respectively. The stimulatory effect of CRF on both parameters is inhibited by the dopaminergic agonist 2-bromo-α-ergocryptine (CB-154). The present data show that CRF is a potent stimulator of peptide secretion in the intermediate lobe of the pituitary gland and suggest a role of this pituitary lobe in the response to stress. In addition, the present data clearly indicate a role of cyclic AMP as mediator of the action of CRF in pars cells.     

Localization of Nitric Oxide-related Substances in the Quail Ovary During Folliculogenesis

In the present study, nitric oxide (NO)-related substances, namely NO synthase (NOS), L-citrulline, cGMP and nitrotyrosine, have been localized in the quail ovary, using NADPH-diaphorase staining and immunohistochemical methods. The results indicate the presence of the NOS isoforms, showing distinct cell-specific distribution patterns in the quail ovary. Inducible NOS is primarily present in leukocytes, endothelial NOS in granulosa cells, and neuronal NOS in nerve cells, oocytes, interstitial cells and granulosa cells of pre-hierarchal follicles and of the germinal disc region of pre-ovulatory follicles. NOS activity, indicated by the presence of L-citrulline, is observed in oocytes, nerve cells, interstitial cells and a few granulosa cells of pre-hierarchal follicles. Detection of accumulated cGMP indicates that granulosa cells of pre-hierarchal and of pre- and post-ovulatory follicles, the theca interna of pre-ovulatory follicles, and oocytes are main targets of NO. Nitrotyrosine, a marker of peroxynitrite activity, is mainly localized in atretic follicles and in post-ovulatory follicles. lt is concluded that the quail ovary possesses a NO/NOS system, and that NO may be considered as a mediator involved in various ovarian processes, including atresia.     

Major impact of hormonal therapy in localized prostate cancer--death can already be an exception

For about 50 years, androgen blockade in prostate cancer has been limited to monotherapy (surgical castration) or high doses of estrogens in patients with advanced disease and bone metastases. The discovery of medical castration with LHRH agonists has led to fundamental changes in the endocrine therapy of prostate cancer. In 1979, the first prostate cancer patient treated with an LHRH agonist received such treatment at the Laval University Medical Center. A long series of studies have clearly demonstrated that medical castration with an LHRH agonist has inhibitory effects on prostate cancer equivalent to those of surgical castration. The much higher acceptability of LHRH agonists has been essential to permit a series of studies in localized disease. Based upon the finding that the testicles and adrenals contribute approximately equal amounts of androgens in the human prostate, the combination of medical (LHRH agonist) or surgical castration associated with a pure antiandrogen (flutamide, nilutamide or bicalutamide) has led to the first demonstration of a prolongation of life in prostate cancer, namely a 10–20% decreased risk of death according to the various metaanalyses of all the studies performed in advanced disease. In analogy with the other types of advanced cancers, the success of combined androgen blockade in metastatic disease is limited by the development of resistance to treatment. To avoid the problem of resistance to treatment while taking advantage of the relative ease of diagnosis of prostate cancer at an “early” stage, the much higher acceptability of LHRH agonists has permitted a series of studies which have demonstrated a major reduction in deaths from prostate cancer ranging from 31% to 87% at 5 years of follow-up in patients with localized or locally advanced prostate cancer. Most importantly, recent data show that the addition of a pure antiandrogen to an LHRH agonist in order to block the androgens made locally in the prostate leads to a 90% long-term control or probable cure of prostate cancer.     

Induction of the avian coelom with associated vitelline blood circulation by Rauber's sickle derived junctional endoblast and its fundamental role in heart formation

In histological sections through chicken blastoderms of different ages we describe the temporospatial relationship between junctional endoblast, the formation of blood islands (appearing first from a peripherally migrating mesoblastic blastema), and the formation of coelomic vesicles developing later in/and from a more superficially extending mesoblastic blastema (coelomic mesoblast). After unilateral removal of the Rauber's sickle-derived junctional endoblast in early streak blastoderms (stage 2-4; Vakaet [1970] Arch Biol 81:387-426) and culture to stage 11 (Hamburger and Hamilton [1951] J Morphol 88:49-92), we observed that the early formation of the coelomic cavity was locally or totally disturbed in the operated area. Besides the simultaneous absence of blood islands, the coelomic vesicles did not form normally. Instead of regularly aligned coelomic vesicles, progressively forming the coelomic cavity by fusion, some voluminous irregular cavities appeared. Thus, the extent of the coelomic cavity was greatly reduced and the operated side was considerably smaller than the unoperated side. Furthermore, in the youngest operated blastoderms the cranial portion of the involved coelomic cavity (hemipericardial cavity) exhibited rudimentary development and usually did not reach the region of the foregut endoderm. This resulted in the absence of the myoepicardium and associated endocardium at this side. In another experiment, after removal of the junctional endoblast at one side of the chicken blastoderm, a fragment of quail junctional endoblast was placed isotopically. This resulted, after further in vitro culture, in the restoration of the formation of coelomic vesicles and accompanying subjacent blood islands in the immediate neighborhood of the apposed quail junctional endoblast. Also, the pericardium and primary heart tube developed normally. Similarly, by using the quail-chicken chimera technique, we demonstrated that the splanchnic mesoderm cells of the pericardium develop in intimate association with the most cranial part of the junctional endoblast (derived from the Rauber's sickle horns). Our experiments indicate that the coelom and, in particular, the pericardium and primary heart tube form progressively (in time and space) under the inductory influence of Rauber's sickle and junctional endoblast.     

Primary production in the deep chlorophyll maximum of the central North Sea

Deep chlorophyll maxima (CM) are commonly observed in the summerstratified North Sea. This feature was studied north of DoggerBank in August and showed high chlorophyll a (Chl a) concentration(     

Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration

Nitrative stress has an important role in microvascular degeneration leading to ischemia in conditions such as diabetic retinopathy and retinopathy of prematurity. Thus far, mediators of nitrative stress have been poorly characterized. We recently described that trans-arachidonic acids are major products of NO(2)(*)-mediated isomerization of arachidonic acid within the cell membrane, but their biological relevance is unknown. Here we show that trans-arachidonic acids are generated in a model of retinal microangiopathy in vivo in a NO(*)-dependent manner. They induce a selective time- and concentration-dependent apoptosis of microvascular endothelial cells in vitro, and result in retinal microvascular degeneration ex vivo and in vivo. These effects are mediated by an upregulation of the antiangiogenic factor thrombospondin-1, independently of classical arachidonic acid metabolism. Our findings provide new insight into the molecular mechanisms of nitrative stress in microvascular injury and suggest new therapeutic avenues in the management of disorders involving nitrative stress, such as ischemic retinopathies and encephalopathies.